维生素A对体外培养小鼠精原干细胞生长增殖的影响

Vitamin A effects on growth and proliferation of mouse spermatogonial stem cells during in vitro culture

背景:维生素A在体内对精原干细胞生长具有重要作用,目前还没有发现在体外培养过程中能够很好促进精原干细胞生长与分化的诱导物质。目的:探讨维生素A对体外培养小鼠精原干细胞生长增殖的影响。方法:无菌收集5～7d龄昆明雄性小鼠双侧睾丸,采用差速贴壁联合非连续性Percoll密度梯度离心法分离纯化精原干细胞无菌取出12～15d龄昆明雄性小鼠双侧睾丸,酶消化法分离纯化Sertoli细胞,贴壁并极化后作为饲养层,将精原干细胞接种在单层Sertoli细胞上。设立2组,实验组向DMEM/F12培养液中加入1g/L维生素A,对照组不添加维生素A。采用酶联仪测定精原干细胞生长增殖情况,流式细胞仪检测精原干细胞生长周期。结果与结论:共培养6,9,12,15d时,实验组精原干细胞吸光度值明显高于对照组(P<0.05或0.01)。随共培养时间的延长,实验组精原干细胞S期染色体含量逐渐增多,然后又逐渐下降,开始另一个分裂周期;与实验组比较,对照组精原干细胞S期染色体含量增长缓慢(P<0.05)。小鼠精原干细胞在体外培养过程中,维生素A可促进其增殖分化。

BACKGROUND： Vitamin A has important effects on growth of spermatogonial stem cells. At present, we have not found an inductive substance of promoting growth and differentiation of spermatogonial stem cells during in vitro culture. OBJECTIVE： To investigate the effect of Vitamin A on growth and proliferation of mouse spermatogonial stem cells in vitro culture. METHODS： Bilateral testis of Kunming male mice aged 5-7 days were sterilely collected. Spermatogonial stem cells were isolated and purified using adherence and noncontinuity Percoll density gradient centrifugation. Bilateral testis was obtained from Kunming male mice aged 12-15 days under sterile conditions. Sertoli cells were isolated and purified by using enzyme digestion method. Following adherence and polarization, Sertoli cells served as feeder layer. The spermatogonial stem cells were seeded on simple-layer Sertoli cells. We set two groups, In the experimental group, 1 g/L Vitamin A was added in the DMEM/F12. In the control group, Vitamin A was not added. Growth and proliferation of spermatogonial stem cells were measured using enzyme linked immunosorbent assay. The cell cycle of spermatogonial stem cells was determined by flow cytometry. RESULTS AND CONCLUSION： At days 6, 9, 12 and 15 following coculture, absorbance value of spermatogonial stem cells in experimental group was faster than control group （P 0.05 or 0.01）. With prolongation of coculture, quantity of chromosome in S phase in spermatogonial stem cells was increased, and then decreased in the experimental group. Another division cycle began. Compared with experimental group, quantity of chromosome in S phase in spermatogonial stem cells was slowly increased in the control group （P 0.05）. During in vitro culture of mouse spermatogonial stem cells, Vitamin A can improve the proliferation and polarization of spermatogonial stem cells.